Enterococci are Gram-positive constituents of the human microflora and play beneficial roles in artisanal cheese production and as probiotics to treat gastroenteritis. Enterococci have emerged as one of the leading causes of nosocomial infections. The problem of enterococcal infection has been aggravated by the emergence of multiple antibiotic resistance that poses a serious challenge to therapeutic intervention. This is an issue of concern in medicine, to the scientific community and the general public. Direction in the search for new therapeutic approaches for treating enterococcal infections will come from a clearer understanding of the factors involved in the pathogenesis of the disease and their regulatory mechanisms. The availability of the E. faecalis genome sequence allowed us to identify the key components of the bacterial regulatory mechanisms carried out by two-component signal transduction systems. The research proposed in this application has the goal of defining the regulatory network of enterococcal signal transduction for the purpose of understanding the mechanisms underlying the physiology of these organisms. This will provide valuable information for the understanding of mechanisms responsible for pathogenicity and antibiotic resistance and thus the identification of molecular targets for therapeutic intervention. We have generated deletion mutants for all response regulators identified in E. faecalis V583. Phenotypes have been identified in some strains, and a role for the FsrA response regulator in controlling biofilm formation through regulation of gelatinase production has been revealed. We propose to: 1) determine the role of two-component systems in virulence through in vivo analyses; 2) determine the role of gelatinase in biofilm; 3) define the genes regulated by two-component signal transduction systems (the regulon) by transcriptional and proteomic approaches; 4) determine the role of the two-component systems in enterococcal physiology by turning ON or OFF the genes encoding response regulatory proteins and analyzing the resulting phenotypes. [unreadable] [unreadable]